Protocol: Rehydrating embryos once fixed and dehydrated…
Posted by Gary McDowell, on 9 July 2010
Does anyone know a protocol for rehydrating embryos once stored in methanol? I’m working with Xenopus laevis embryos which have been fixed using formaldehyde. Some lab wisdom passed down is “wash into a 50:50 solution of 1xPBS and methanol; then into 1xPBS; then into methanol again (which we’re a bit confused by); then into 50:50 PBS: methanol; and finally 1xPBS”.
Any help would be much appreciated!
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Hi Gary.
We used to fix mouse embryos (both pre- and post-implantation) in PFA then methanol. After your standard PFA fix, wash in PBS then pass them through 25%, 50%, 75% then 100% methanol (in PBS, 5mins each step). To rehydrate, just do the reverse: 75%, 50%, 25% then back into PBS. This worked fine for me and others in the lab (the embryos do get a little sticky while in the methanol, though!).
Hope this helps
Thanks Seema!
I’ve just carried it out as you suggested – my main problem was that I coinject embryos with beta-galactosidase and then stain to check for injection using X-gal, but had forgotten to do this before dehydrating embryos.
The staining is taking much longer but does seem to be working – thank you!
Gary – what Seema suggested is exactly what we do with Xenoups laevis embyros! So it should work fine! (ps only fix in formaldehyde ((memfa)) for one hour when doing x-gal staining) Ruth